The long term goal of this R21 project is to develop tools for imaging lymphatic vessels (LVs) in living mice. Lymphatic vessels drain interstitial fluid by means of valve-like openings, they transport lipids and they participate in the immune system. They transport antigen and antigen presenting cells to lymph nodes and cells from the lymph node to the circulation. When lymphatic vessels are disrupted, lymphedema, the accumulation of lymph in the interstitial tissues, occurs. Immunological functions are thwarted. Lymphangiogenesis occurs in inflammation at the site of immunization and in lymph nodes (secondary lymphoid organs) where there is an interaction between LVs and high endothelial venules (HEVs). Lymphangiogenesis occurs in chronic inflammation in lymphoid accumulations in ectopic sites, called tertiary lymphoid tissues (TLOs) that have many characteristics of lymph nodes. Mice transgenic for the rat insulin promoter (RIP) driving lymphotoxin-1 (RIPLT1) exhibit such TLOs in the pancreas. Techniques that have been used to develop mice whose HEVs can be imaged via green fluorescent protein will be used here to develop mice whose LVs can be similarly imaged via a red fluorescent protein. To attain this goal, the following specific aims will be accomplished: 1. To develop transgenic mice whose lymphatic vessels express fluorescent proteins. The pCLASPER recombineering technology will be used to isolate LV genes (prox1, lyve-1, or podoplanin), insert red fluorescent reporter genes, such as tdTomato, and produce transgenic mice. 2. To evaluate lymphatic reporter transgenic mice for fidelity of expression. Mice transgenic for lymphatic vessel reporter constructs will be analyzed for co- expression of endogenous LV genes and transgenes in several tissues in the steady state, in lymph nodes after immunization, and in TLOs in RIPLT1 mice. 3. To evaluate LVs in living mice. Multiphoton in vivo microscopy will be used to evaluate fluorescent LVs. Interactions between (green) high endothelial venules and (red) lymphatic vessels in the lymph nodes of immunized mice will be analyzed in real time. A dynamic understanding of lymphangiogenesis and LV function will provide therapeutic insight into lymphatic function and dysfunction in lymphedema. The development of imaging tools for lymphatic vessels will provide insight into how LVs and HEVs are regulated, maintained, and cooperate during an immune response and provide insight into lymphangiogenesis in inflammation, transplantation, autoimmunity, infectious diseases, and cancer. PUBLIC HEALTH RELEVANCE: A thorough understanding of lymphangiogenesis and lymphatic vessel function will provide therapeutic insight into lymphatic function and dysfunction in conditions such as lymphedema. Imaging tools for lymphatic vessels will provide insight into how this vascular system is maintained and regulated during an immune response and provide insight into lymphangiogenesis in inflammation, transplantation, autoimmunity, infectious diseases, and cancer. )